Designing effective strategies to combat HIV-1 requires structural knowledge of how antibodies (Abs) recognize HIV envelope proteins and how they are used by the immune system to eliminate viruses and virally-infected cells. Until recently, only a small number of broadly neutralizing antibodies (bNAbs) against HIV-1 had been characterized and the immunological basis for their breadth and potency remains poorly understood. The limited availability of bNAbs was resolved by new methods developed by Dr. Nussenzweig for HIV Ab isolation from the B cells of HIV-infected individuals. Several of these Abs, including NIH45-46, show superior potency and breadth to VRCOl, previously the best bNAb. We collaborated with Dr. Nussenzweig to solve the structure of NIH45-46 bound to gpl 20, and used structure-based design to increase its potency at least 10-fold, making NIH45-46'^^''^ the best anti-HIV bNAb available as of its publication in 2011; we have now made an even more effective version (45-46m2) that neutralizes up to 97% of HIV strains. We will now collaborate with Drs. Nussenzweig to determine their mechanisms of action in animal models. The Ravetch laboratory has developed amino acid and glycan modifications of IgG Fc regions that optimize binding to activating Fc receptors, which will be introduced into designed bNAbs to increase their effector functions. In collaboration with Drs. Nussenzweig and Ravetch, we propose to determine the structural correlates of (i) broad/potent neutralization by the variable Fabs of bNAbs, and (ii) improved effector functions mediated by Fc regions. Information gleaned from these structures is directly relevant to reverse vaccination involving gene therapy to deliver improved bNAbs and also required for immunogen design. This collaboration presents the opportunity to merge structural biology with in vitro and in vivo efficacy evaluations of new bNAbs against HIV, which will be facilitated by a set of scientific cores that will permit all three laboratories to quickly and accurately measure neutralization potencies and binding profiles of bNAbs (Core A, Automated Cell/Biochemical Assays Core), express large numbers of different bNAbs and HIV proteins (Core B, Protein Expression Core), and conduct experiments with HIV-infected humanized mice (Core C, Animal Services Core). The following specific aims will be pursued collaboratively: 1) Solve crystal structures of bNAb Fabs and bNAb Fab-gp120 complexes to define essential features of broad/potent HIV neutralization. 2) Solve crystal structures of IgG Fc regions and Fc/Fc receptor complexes to determine the structural effects of effector function-altering amino acid and glycan changes. 3) Produce natural antibody variants with enhanced activity in neutralization and mediating effector functions. 4) Use knowledge gained from these structural studies to design immunogens to elicit bNAbs. RELEVANCE (See instructions): HIV/AIDS remains a critical threat to global public health. At least 60 million people have been infected with HIV. Although anti-retroviral drugs have been effective in the developed world, a vaccine and/or new methods to prevent infections are needed in the developing world. We will systematically analyze the structures of anti-HIV antibodies with the goal of understanding their protective mechanisms and routes of viral resistance, information that will be used to improve their efficacy and to design immunogens for vaccines.